miR‐2861 as novel HDAC5 inhibitor in CHO cells enhances productivity while maintaining product quality

Abstract

Histone deacetylase (HDAC) inhibitors have been exploited for years to improve recombinant protein expression in mammalian production cells. However, global HDAC inhibition is associated with negative effects on various cellular processes. microRNAs (miRNAs) have been shown to regulate gene expression in almost all eukaryotic cell types by controlling entire cellular pathways. Since miRNAs recently have gained much attention as next-generation cell engineering tool to improve Chinese hamster ovary (CHO) cell factories, we were interested if miRNAs are able to specifically repress HDAC expression in CHO cells to circumvent limitations of unspecific HDAC inhibition. We discovered a novel miRNA in CHO cells, miR-2861, which was shown to enhance productivity in various recombinant CHO cell lines. Furthermore, we demonstrate that miR-2861 might post-transcriptionally regulate HDAC5 in CHO cells. Intriguingly, siRNA-mediated HDAC5 suppression could be demonstrated to phenocopy pro-productive effects of miR-2861 in CHO cells. This supports the notion that miRNA-induced inhibition of HDAC5 may contribute to productivity enhancing effects of miR-2861. Furthermore, since product quality is fundamental to safety and functionality of biologics, we examined the effect of HDAC inhibition on critical product quality attributes. In contrast to unspecific HDAC inhibition using VPA, enforced expression of miR-2861 did not negatively influence antibody aggregation or N-glycosylation. Our findings highlight the superiority of miRNA-mediated inhibition of specific HDACs and present miR-2861 as novel cell engineering tool for improving CHO manufacturing cells.

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

“…Furthermore, individual miRNAs are capable of regulating entire cellular pathways (Fischer et al, ; Hackl et al, ), and thus might have a substantial impact on CHO cell phenotypes. Currently available literature on miRNA research in CHO cells mostly focusses on the identification of miRNAs to improve recombinant protein production (Emmerling et al, ; Fischer et al, , ; Jadhav et al, ; Kelly et al, ; Loh et al, ), or cell growth (Barron et al, ; Druz et al, ; Sanchez et al, ), while other studies were dedicated to basic principles of miRNA biogenesis and function (Diendorfer et al, ; Fischer et al, ,; Hackl et al, , ; Hernandez Bort et al, ). We have previously identified miR‐557 to increase productivity of a recombinant easy‐to‐express mAb in CHO cells (Strotbek et al, ).…”

miR‐CATCH Identifies Biologically Active miRNA Regulators of the Pro‐Survival Gene XIAP, in Chinese Hamster Ovary Cells

“…To navigate around this, microRNA‐based engineering has an added advantage owing to the non‐coding nature of these regulatory RNAs so as to not exert a translational burden on the CHO producer cell . Stable miRNA interference in a variety of CHO cell lines has generated improved phenotypes including higher product yields such as in the case of miR‐466h, miR‐2861, and miR‐557 . Given the limitations in miRNA target prediction, it is critical to identify bona fide miRNA regulators for the species, cell type, and gene under investigation as a means to increase the potential for a positive outcome.…”

“…Druz and colleagues reported the induction of apoptosis resistance in a CHO cell line stably engineered to be depleted of miR‐466h‐5p with an associated increase in the levels of anti‐apoptotic genes such as birc6 and bcl2l2 . Other studies across various research groups have further supported the utility of miRNA as cell engineering candidates including miR‐23, miR‐2861, miR‐17, and miR‐557 . In these instances, single miRNA engineering mediated a beneficial CHO cell phenotype via a multitude of molecular networks under regulation.…”

Identifying HIPK1 as Target of miR‐22‐3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen

“…The search for the specific affected genes was done by using microarrays, high‐throughput screening, next generation sequencing, and/or quantitative reverse transcription PCR (qRT‐PCR) . While these studies did not describe the specific mechanism by which the microRNA acted, they identified genes and pathways involved in apoptosis, HDAC5 modulation, and the ubiquitin pathway …”